TW201909926A - B7H3 antibody-drug conjugate and its medical use - Google Patents

Abstract

The present invention relates to B7H3 antibody-cytotoxic drug conjugate and pharmaceutical use thereof. In particular, the present invention relates to a B7H3 antibody-cytotoxic drug conjugate, a pharmaceutically acceptable salt or solvate thereof. And a pharmaceutical composition comprising said conjugate, or a pharmaceutically acceptable salt or solvate thereof, and their use in the preparation of a medicament for the treatment of B7H3-mediated disease or symptom, especially their use as anti-cancer agents.

Description

   B7H3 antibody-drug conjugate and its medical use   

The present invention relates to a B7H3 antibody-drug conjugate and its application in medicine. Further, the present invention relates to a B7H3 antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, and comprises the aforementioned coupling Or a pharmaceutically acceptable salt or solvent compound of the pharmaceutical composition, and its use in the preparation of a medicament for the treatment of a B7H3-mediated disease or disorder; especially its use in the preparation of an anticancer medicament.

T cell-mediated immune response plays an extremely important role in the body's anti-tumor process. T cell activation and proliferation require not only the antigen signal recognized by TCR, but also the second signal provided by the co-stimulatory molecule. B7 family molecules belong to the co-stimulatory molecule immunoglobulin superfamily. More and more studies show that this family of molecules play an important regulatory role in the body's normal immune function and pathological state.

B7H3 is a member of the B7 family and belongs to a type I transmembrane protein. It contains a signal peptide at the amine end, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region, and One cytoplasmic tail region containing 45 amino acids (Tissue Antigens. 2007 Aug; 70 (2): 96-104). At present, there are two main types of spliceosome in B7H3, B7H3a and B7H3b. The extracellular segment of B7H3a is composed of two IgV-IgC immunoglobulin domains, also known as 2IgB7H3, while the extracellular segment of B7H3b is composed of four IgV-IgC-IgV-IgC immunoglobulin domains, also known as 4IgB7H3.

B7H3 protein is not expressed in normal tissues or cells or is extremely low expressed, but is highly expressed in a variety of tumor tissues, and is closely related to tumor progression, patient survival and prognosis. Clinically, B7H3 has been reported in many cancer types, especially in non-small cell lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. Expression (LungCancer. 2009 Nov; 66 (2): 245-249; ClinCancerRes. 2008 Aug15; 14 (16): 5150-5157). In addition, there are reports in the literature that in prostate cancer, the expression intensity of B7H3 is positively correlated with clinical pathological malignancies (such as tumor volume, extraprostatic invasion, or Gleason score), and also related to cancer progression (CancerRes. 2007Aug15; 67 (16) : 7893-7900). Similarly, in glioblastoma multiforme, B7H3 expression was negatively correlated with event-free survival, and in pancreatic cancer, B7H3 expression was correlated with lymph node metastasis and pathological progression. Therefore, B7H3 is considered as a new tumor marker and potential therapeutic target.

At present, there are treatment strategies targeting B7H3 targets for preclinical studies. For example, antibodies targeting mouse B7H3 can enhance invasive CD8-positive T cells in tumors and inhibit tumor growth (ModPathol. 2010 Aug; 23 ( 8): 1104-1112). In addition, the WO2008 / 066691 patent shows that antibodies that recognize the B7H3 variant B7H3a will exhibit antitumor effects on adenocarcinoma in vivo. In clinical research, a mouse-derived B7H3 antibody coupled with radioactive I ¹³¹ can significantly inhibit the growth of neuroblastoma in patients [JNeufooocol97 (3): 409-18 (2010]. However, the current research projects are all It is a humanized antibody that has been humanized and transformed by a mouse-derived antibody. The humanized antibody has a relatively high immunogenicity problem during immunization, which is a disadvantageous factor in human applications.

Phage display technology (phage display technology) is a fusion expression of a foreign protein or polypeptide and a phage coat protein, thereby expressing the foreign protein on the surface of the phage. The phage antibody library is an antibody library created by combining phage display technology, PCR amplification technology, and protein expression technology with comprehensive technical means.

The biggest advantage of the phage antibody library is to prepare fully humanized antibodies without in vivo immunization and simulating the three processes of antibody production in vivo. In addition, the phage antibody library also has the following advantages: ① The genotype and phenotype are unified. In addition, the experimental method is simple and fast. The traditional method of antibody production by fusion tumor technology takes several months, while the antibody library technology only takes a few weeks. ② It expresses completely human-derived antibodies, and has a small molecular weight. It is mainly expressed in the form of active fragments Fab and scFV. Compared with intact antibodies, it has obvious advantages in tissue penetration. ③ The screening capacity is large. The fusion tumor technology is screening in thousands of pure strains. The antibody library technology can select millions or even billions of molecules. The more types of antibodies are screened. ④ It has a wide range of uses and uses a prokaryotic expression system, which has more obvious advantages when it is produced on a large scale (CurrOpinBiotechnol. 2002 Dec; 13 (6): 598-602; Immunotechnology, 2013, 48 (13): 63-73).

Antibody-drug conjugates (antibodydrugconjugate (ADC) link a single antibody or antibody fragment to a biologically active cytotoxin through a stable chemical linker compound, making full use of the specificity of antibody binding to normal cell and tumor cell surface antigens And the high efficiency of cytotoxic substances, while avoiding the former's low efficacy and the latter's excessive side effects. This means that, compared with traditional chemotherapy drugs, antibody-drug conjugates can bind tumor cells more accurately and reduce the impact on normal cells.

At present, many ADC drugs have been used in clinical or clinical research, such as Kadcyla, which is an ADC drug that targets Her2 to trastuzumab and DM1. At the same time, there are also patent reports of antibodies and ADC drugs targeting B7H3, such as WO2008100934, WO2012147713, WO2014061277, WO2015184203, WO2016044383. However, there are still no B7H3 target ADC drugs on the market or used in clinical treatment research. Therefore, the development of new B7H3 target ADC drugs has broad prospects.

An object of the present invention is to provide an ADC drug coupled with a cytotoxic substance by a monoclonal antibody that binds to an amino acid sequence or a three-dimensional structure of the extracellular region of B7H3. By screening a highly active and stable anti-human B7H3 fully human antibody ADC drug, a therapeutic agent using the antibody ADC drug as an active ingredient is provided.

The invention provides an antibody-drug conjugate represented by the general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof: Ab- (L ₂ -L ₁ -D) _y (A) wherein: D is a cell Toxic drugs; L ₁ and L ₂ are linker units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; y may be a decimal or an integer; Ab is a B7H3 antibody or an antigen-binding fragment thereof, It binds to human B7H3, and the B7H3 antibody or antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment thereof selected from any one of (a) to (c) below: (a) a monoclonal antibody comprising 1 Or more amino acid sequences selected from the following CDR region sequences or having at least 95% sequence identity with them: the antibody heavy chain variable region HCDR region sequence: as shown in SEQ ID NOs: 10, 11 and 12 amino acid sequences And the sequence of the antibody light chain variable region LCDR region: as shown in the amino acid sequences of SEQ ID NOs: 13, 14, and 15; (b) a monoclonal antibody comprising one or more CDR regions selected from Sequence or an amino acid sequence having at least 95% sequence identity with it: the antibody heavy chain variable region HCDR region sequence: as shown in SEQ ID NO: 16, 17, and 18 amino acid sequences; and the antibody light chain may Region LCDR region sequence: as shown in SEQ ID NO: 19, 20 and 21 amino acid sequences; (c) a monoclonal antibody comprising one or more CDR region sequences selected from the following or having at least 95% of the sequence thereof Identical amino acid sequence: antibody heavy chain variable region HCDR region sequence: as shown in SEQ ID NO: 30, 31 and 32 amino acid sequence; and antibody light chain variable region LCDR region sequence: as SEQ ID NO : 33, 34 and 35 amino acid sequences are shown.

In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (A), wherein the B7H3 antibody or antigen-binding fragment thereof is selected from the following ( a) a monoclonal antibody or an antigen-binding fragment thereof according to any one of (a) to (c): (a) a monoclonal antibody comprising a variable antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 10, 11, and 12 respectively; HCDR1, HCDR2, HCDR3 of the region; and LCDR1, LCDR2, LCDR3 of the light chain variable region of the antibody as shown in the amino acid sequences of SEQ ID NOs: 13, 14, and 15; (b) a monoclonal antibody comprising, ID NO: HCDR1, HCDR2, HCDR3 of the heavy chain variable region of the antibody shown in the amino acid sequence of 16, 17, and 18; and light chain variable region of the antibody shown in the amino acid sequence of SEQ ID NO: 19, 20, and 21 LCDR1, LCDR2, LCDR3; (c) a monoclonal antibody comprising HCDR1, HCDR2, HCDR3 of the variable region of the antibody heavy chain as shown in the amino acid sequences of SEQ ID NOs: 30, 31 and 32, respectively; ID NOs: LCDR1, LCDR2, LCDR3 of the light chain variable region of the antibody shown by the amino acid sequences of 33, 34 and 35.

In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (A), Ab- (L ₂ -L ₁ -D) _y (A ): D is a cytotoxic drug; L ₁ and L ₂ are linker units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is a B7H3 antibody as defined above or The antigen-binding fragment competes for a monoclonal antibody or an antigen-binding fragment thereof that binds human B7H3.

In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein the Ab is a recombinant antibody.

In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein the Ab is a recombinant antibody of human origin or an antigen-binding fragment thereof.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the sequences of the light and heavy chain FR regions on the light and heavy chain variable regions of the Ab are respectively derived from Human germline light and heavy chain sequences or mutant sequences thereof.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the constant region of the Ab includes a heavy chain derived from human IgG1, IgG2, IgG3, or IgG4 or a variant thereof The constant region is preferably a human IgG1 heavy chain constant region; and the light chain constant region derived from a human-derived kappa, lambda chain, or a variant thereof, preferably a human kappa-chain light chain constant region.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the Ab contains SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36 or SEQ ID NO : 37 heavy chain variable region or a variant thereof; the variant is a heavy chain variable region shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36 or SEQ ID NO: 37 The region sequence has a sequence of 1 to 10 amino acid substitutions.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the Ab contains SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38, or SEQ ID NO : The light chain variable region shown in 39 or a variant thereof; the variant is a light chain variable shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38, or SEQ ID NO: 39 The region sequence has a sequence of 1 to 10 amino acid substitutions.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the Ab contains a monoclonal antibody or an antigen binding agent thereof selected from any one of (1) to (7) below Fragments: (1) a monoclonal antibody comprising the heavy chain variable region of the antibody shown in SEQ ID NO: 6; and a light chain variable region of the antibody shown in SEQ ID NO: 7; (2) a monoclonal antibody comprising The antibody heavy chain variable region shown in SEQ ID NO: 8; and the antibody light chain variable region shown in SEQ ID NO: 9; (3) a monoclonal antibody comprising the antibody heavy chain shown in SEQ ID NO: 28 A variable region; and the light chain variable region of the antibody shown in SEQ ID NO: 29; (4) a monoclonal antibody comprising the antibody heavy chain variable region shown in SEQ ID NO: 36; and SEQ ID NO: 38 (5) a monoclonal antibody comprising the antibody heavy chain variable region shown in SEQ ID NO: 36; and the antibody light chain variable region shown in SEQ ID NO: 39; (6) A monoclonal antibody comprising the heavy chain variable region of the antibody shown in SEQ ID NO: 37; and a light chain variable region of the antibody shown in SEQ ID NO: 38; (7) a monoclonal antibody comprising SEQ ID NO: 37 The heavy chain variable region of the antibody shown; and the antibody of SEQ ID NO: 39 Chain variable region.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the Ab is a full-length antibody, which further includes a human antibody constant region; wherein the full-length antibody is selected from the group consisting of an h1702 antibody, It is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO: 22 and the light chain sequence shown in SEQ ID NO: 23, and the h1703 antibody is a heavy chain sequence shown in SEQ ID NO: 24 and SEQ A full-length antibody composed of the light chain sequence shown in ID NO: 25 and an h1702-DS antibody, which is a full-length antibody composed of the heavy chain sequence shown in SEQ ID NO: 22 and the light chain sequence shown in SEQ ID NO: 26 Or h1704-3 antibody, which is a full-length antibody composed of the heavy chain sequence shown in SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 41.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the antigen-binding fragment is selected from Fab, Fab ', F (ab') 2, and single-chain antibody (scFv) , A dimerized V region (diabody), a disulfide stabilized V region (dsFv), and an antigen-binding fragment of a CDR-containing peptide.

In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein the cytotoxic drug is selected from the group consisting of a toxin, a chemotherapeutic drug, an antibiotic, a radioisotope, and a nucleolytic enzyme.

In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein the cytotoxic drug is selected from DM1, DM3, DM4, MMAF and MMAE.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A), wherein the cytotoxic drug is selected from:

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A) is a compound represented by Formula I or a pharmaceutically acceptable salt or solvent compound thereof, Wherein: L ₁ and L ₂ are linker units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is a B7H3 antibody or an antigen-binding fragment thereof as defined above, or as described above A defined B7H3 antibody or antigen-binding fragment thereof competes for a monoclonal antibody or antigen-binding fragment thereof that binds human B7H3.

In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein L _{2 is represented by the} following general formula L ₂ : Wherein X _{1 is} selected from a hydrogen atom, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl; X _{2 is} selected from alkyl, cycloalkyl and heterocyclic; m is an integer selected from 0 to 5 , Preferably 1, 2 or 3; S is a sulfur atom.

In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (A), wherein L _{1 is represented by the} following general formula (L ₁ ): Wherein X ₃ is an alkyl group, the alkyl group is further substituted with a substituent selected from a halogen, a hydroxyl group, and a cyano group if necessary; n is an integer selected from 0 to 5, preferably 1, 2 or 3.

In a preferred embodiment, the antibody-drug conjugate shown by the general formula (A), wherein the cytotoxic drug is connected to the linker L1 to obtain a compound:

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A) is an antibody-drug conjugate represented by the general formula (II): Wherein L ₂ is a linker unit; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is a B7H3 antibody or an antigen-binding fragment thereof as defined above, or B7H3 as defined above The antibody or antigen-binding fragment thereof competes for a monoclonal antibody or antigen-binding fragment thereof that binds human B7H3.

In a preferred embodiment, the antibody-drug conjugate represented by the general formula (A) is an antibody-drug conjugate represented by the general formula (III): Among them, L ₁ is a linker unit; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is a B7H3 antibody or an antigen-binding fragment thereof as defined above, or B7H3 as defined above The antibody or antigen-binding fragment thereof competes for a monoclonal antibody or antigen-binding fragment thereof that binds human B7H3.

In a preferred embodiment, the antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, as shown by the general formula (A) includes, but is not limited to: .

The invention further relates to a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (A) of the invention, and one or more pharmaceutically acceptable excipients Agent, diluent or carrier.

The present invention further relates to an antibody-drug conjugate represented by the general formula (A), or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition containing the same, which is prepared for use in treating human B7H3-positive cells. The use in a medicine for a disease; preferably, the use is for preparing a medicine for treating B7H3 overexpressing cancer.

The present invention further relates to the use of an antibody-drug conjugate represented by the general formula (A), or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition comprising the same in the manufacture of a medicament for treating a disease, The disease is selected from the group consisting of the treatment of gliomas (non-limiting examples are human brain astrocyte tumors), human pharyngeal carcinoma, adrenal tumors, AIDS-related cancer, acinar soft tissue sarcoma, astrocytoma, bladder cancer, Bone cancer, brain and spinal cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal cell carcinoma, clear cell cancer, colon cancer, colorectal cancer, connective tissue Proliferative small round cell tumors, ependymal cell tumors, Ewing tumors, extra-bone myxoid chondrosarcoma, osteogenesis, osteofibrosis, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, Head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipoma tumor, liver cancer, lymphoma, lung cancer, neuroblastoma, black Tumors, meningiomas, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, children Cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdomyosarcoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, slippery Mesosarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, and uterine cancer.

The present invention further relates to a method for treating a disease, the method comprising administering to a patient in need thereof a therapeutically effective dose of the antibody-drug conjugate represented by the general formula (A), or a pharmaceutically acceptable salt or solvent compound thereof Or a medicinal composition comprising the same, the disease is selected from the group consisting of human brain astrocyte tumor, human pharyngocarcinoma, adrenal tumor, AIDS-related cancer, acinar soft tissue sarcoma, astrocytoma, bladder cancer, and bone cancer , Brain and spinal cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal cell carcinoma, clear cell cancer, colon cancer, colorectal cancer, connective tissue proliferative Small round cell tumor, ependymal cell tumor, Ewing tumor, extra-bone myxoid chondrosarcoma, bone fiber hypoplasia, osteofibroplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer , Hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, neuroblastoma Melanoma, meningiomas, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, children Cancer, peripheral schwannoma, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdomyosarcoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, slippery Mesosarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, and uterine cancer.

Figure 1 shows the binding ability of antibodies to U87MG cells; Figure 2 shows the endocytosis effect of different antibodies on U87MG cells; Figure 3 shows the endocytosis effect of different ADCh1702-3024 and h1703-3024 on U87MG cells of the present invention; Figure 4 shows the endocytosis effect of different ADCh1702-cys-3024, h1702DS-cys-3024 and h1704-3-cys-3024 of the present invention on U87MG cells; Figure 5 shows h1702-3024 on nude mice Efficacy of U87MG xenografts; Figure 6 shows the effect of h1702-3024 on the weight of U87MG nude mice.

Figure 7 shows the stability results of h1702DS-cys-3024 and h1704-3-cys-3024.

I. Terminology

To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are described in J. biol. Chem, 243, p3558 (1968).

The "antibody" in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond. The composition and arrangement of amino acids in the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa chains or lambda chains by different constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.

The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions. The sequence from amine end to carboxy end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment according to the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in terms of number and position, or conform to Kabat and chothia. Numbering rules (HCDR1).

The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, and preferably humanized antibodies.

The term "murine antibody" in the present invention is a monoclonal antibody to human B7H3 prepared according to the knowledge and skill in the art. The test subject is injected with B7H3 antigen during preparation, and then a fusion tumor expressing an antibody having a desired sequence or functional characteristics is isolated. In a preferred embodiment of the present invention, the mouse-derived B7H3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived κ, lambda chain or a variant thereof, or further include a mouse-derived IgG1, IgG2, The heavy chain constant region of IgG3 or a variant thereof.

The term "recombinant antibody" includes "chimeric antibodies", "humanized antibodies" and "fully human antibodies".

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody. To establish a chimeric antibody, first establish a fusion tumor that secretes a mouse-specific monoclonal antibody, and then select the variable region gene from the mouse fusion tumor cell, and then select the constant region gene of the human antibody as needed to change the mouse variable The region gene and the human constant region gene are linked into a chimeric gene and inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the antibody light chain of the B7H3 chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the B7H3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or an amino acid is used Mutations (such as YTE mutations or back mutations) of IgG1, IgG2 or IgG4 variants.

The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibodies Antibodies produced in framework sequences. It can overcome the heterogeneous response induced by the chimeric antibody because it carries a large amount of mouse protein components. Such framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc. Human, 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence may be subjected to minimal reverse mutation or back mutation to maintain the activity. The humanized antibody of the present invention also includes a humanized antibody that further undergoes affinity maturation of CDRs by phage display. In a preferred embodiment of the present invention, the mouse CDR sequence in the B7H3 humanized antibody is selected from the group consisting of SEQ ID NO: 8-13 or 14-19; the human antibody variable region framework is designed and selected, wherein the antibody The heavy chain FR region sequence on the variable region of the weight chain is derived from the combined sequence of the human germline heavy chain IGHV1-18 * 01 and hjh4.1 or the combined sequence of IGHV3-7 * 01 and hjh4.1; wherein the antibody is light The light chain FR region sequence on the chain variable region is derived from the combined sequence of the human germline light chain IGKV1-33 * 01 and hjk4.1 or the combined sequence of IGKV1-39 * 01 and hjk2.1. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region can be subjected to a minimum of reverse mutations to maintain the activity.

The transplantation of CDRs may result in a decrease in the affinity of the B7H3 antibody or antigen-binding fragment thereof produced for the antigen due to the framework residues in contact with the antigen. Such interactions can be the result of highly mutated somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of a humanized antibody. The amino acid residues involved in antigen binding from a non-human B7H3 antibody or its antigen-binding fragment can be identified by examining the sequence and structure of the variable region of a mouse monoclonal antibody. Residues in the CDR donor framework that differ from the germline can be considered to be related. If the closest germline cannot be determined, the sequence can be compared to the consensus of a subtype consensus sequence or a murine sequence with a high percentage of similarity. Rare framework residues are thought to be the result of high somatic mutations, and thus play an important role in binding.

The term "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable and constant regions of the antibody are of human origin, removing immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. Relevant technologies for the preparation of fully human antibodies include: human fusion tumor technology, EBV transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology (transgenic mouse), and single B cell antibody preparation technology. The "fully human antibody" in the present invention uses phage display technology to obtain an antibody variable region, which can be further recombined with the antibody constant region to obtain a complete antibody.

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, B7H3). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of the antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment composed of VL, VH, CL, and CH1 domains; (ii) a F (ab ') ₂ fragment, including a borrow A bivalent fragment of two Fab fragments connected by a disulfide bridge on the hinge region, (iii) an Fd fragment composed of VH and CH1 domains; (iv) an Fv composed of one-armed VH and VL domains Fragments; (v) single domain or dAb fragments (Ward et al. (1989) Nature 341: 544-546), which consists of VH domains; and (vi) isolated complementarity determining regions (CDR) or (vii) A combination of two or more separate CDRs, optionally connected by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to link them via a synthetic linker, thereby enabling them to produce a single unit where the VL and VH regions are paired to form a monovalent molecule Protein chains (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883 ). Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using general techniques known to those skilled in the art, and the fragments are functionally screened in the same manner as for intact antibodies. Antigen-binding moieties can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

The antigen-binding fragments of the present invention include Fab, F (ab ') 2, Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-bonded V region (dsFv), CDR peptides, etc.

Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (amino acid residue that cleaves the 224 position of the H chain), in which the H chain N Approximately half of the ends and the entire L chain are held together by disulfide bonds.

The Fab of the present invention can be produced by treating a monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain. In addition, the Fab can be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab.

F (ab ') 2 is obtained by digesting the lower part of two disulfide bonds in the IgG hinge region with the enzyme pepsin and has a molecular weight of about 100,000 and has antigen binding activity and contains two Fab regions connected at the hinge positions. Antibody fragments.

F (ab ') 2 of the present invention can be produced by treating a monoclonal antibody of the present invention that specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with pepsin. Further, the F (ab ') 2 can be produced by connecting a Fab' described below with a thioether bond or a disulfide bond.

Fab 'is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity obtained by cutting a disulfide bond in the hinge region of F (ab') 2 described above. The Fab 'of the present invention can be produced by treating F (ab') 2 of the present invention that specifically recognizes B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.

In addition, the Fab 'can be produced by inserting the DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" is meant to include an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker ) Molecule. Such scFv molecules may have a general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example using 1 to 4 repeating variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444- 6448). Other linkers useful in the present invention are Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

The scFv of the present invention can be produced by the following steps: obtaining cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes human B7H3 of the present invention and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs an encoding scFv DNA, insert this DNA into a prokaryotic or eukaryotic expression vector, and then introduce this expression vector into a prokaryotic or eukaryotic organism to express scFv.

Diabodies are antibody fragments in which scFv is dimerized, and are antibody fragments with bivalent antigen-binding activity. In a bivalent antigen-binding activity, the two antigens may be the same or different.

The diabody of the present invention can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs ScFv-encoding DNA such that the amino acid sequence of the peptide linker is 8 residues or less, insert the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introduce the expression vector into a prokaryotic or Eukaryotes express diabody.

dsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue substituted with a cysteine residue can be selected according to a known method (Protein Engineering, 7,697 (1994)) based on the three-dimensional structure prediction of the antibody.

The dsFv of the present invention can be produced by the following steps: obtaining cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes human B7H3 of the present invention and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs DNA encoding dsFv is inserted into a prokaryotic or eukaryotic expression vector, and the expression vector is introduced into a prokaryote or eukaryote to express dsFv.

A CDR-containing peptide is constructed by including one or more regions in a CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.

The CDR-containing peptides of the present invention can be produced by the steps of constructing the CDRs of the VH and VL of the monoclonal antibodies of the present invention that specifically recognize human B7H3 and bind to the amino acid sequence of the extracellular region or its three-dimensional structure Encoding DNA, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the peptide. The CDR-containing peptide can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDRs applies only to LCDR1, LCDR2, and LCDR3 (CDRL1, CDRL2, CDRL3 or L1, L2, L3) of the light chain variable domain, and HCDR2 and HCDR3 of the variable domain of the heavy chain (CDRH2, CDRH3 or H2, H3).

The term "antibody framework" as used herein refers to a part of a variable domain VL or VH, which serves as a scaffold for the antigen-binding loop (CDR) of the variable domain. In essence, it is a variable domain without a CDR.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a B7H3 molecule). An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 continuous or discontinuous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecula r Biology, Vol. 66, G.E. Morris, Ed. (1996).

The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, antibodies bind with an affinity (KD) of less than about 10 ^-7 M, such as about less than 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less.

The term "KD" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Generally, antibodies of the invention bind to B7H3 with a dissociation equilibrium constant (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less, for example, using surface plasmons Volume resonance (SPR) technology was measured in a BIACORE instrument.

The term "a monoclonal antibody that competes with a B7H3 antibody for binding to human B7H3" refers to a competition with a monoclonal antibody of the invention for recognition of the same epitope (also known as an epitope) or a portion of the same epitope on the extracellular region of human B7H3 And an antibody that binds to the epitope. An antibody that binds to the same epitope as the B7H3 antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human B7H3 recognized by the B7H3 antibody of the present invention.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operatively linked to the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication) or can be integrated into the host cell's genome after introduction into the host cell, thereby accompanying The host genome is replicated together (eg, non-episomal mammalian vectors).

Methods of producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, a mouse can be immunized with human B7H3 or a fragment thereof. The obtained antibody can be coated, purified, and can be subjected to amino acid sequencing by a general method. The antigen-binding fragment can also be prepared by a general method. The antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human-derived FR regions to a CDR region of non-human origin. Human FR germline sequences can be obtained by comparing the IMGT human antibody variable region germline gene database and MOE software from the website of Im Muno Gene Tics (IMGT) http://imgt.cines.fr, or from the immunoglobulin journal , 2001 ISBN012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Easily transformed bacteria include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacilillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NSO cells.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by general methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminal site in the Fc region. Stable pure strains were obtained by expressing antibodies that specifically bind to human B7H3. Positive pure strains were expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium in which the antibody is secreted can be purified by a general technique. For example, use an A or GSepharoseFF column with adjusted buffer for purification. Non-specifically bound components are washed away. The bound antibody was further extracted by pH gradient method, and antibody fragments were detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using a general method. Soluble mixtures and polymers can also be removed by common methods, such as molecular sieves, ion exchange. The resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, recipients Contact with a subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods. Treatment of cells includes contact of the reagent with the cells, and contact of the reagent with the fluid, wherein the fluid is in contact with the cells. "Administering" and "treating" also mean in vitro and ex vivo treatment of, for example, cells by an agent, diagnostic, combination composition, or by another cell. "Treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.

"Treatment" means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, that the patient has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the development of such symptoms to any clinically unmeasurable degree. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") that is effective in alleviating the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. It can be assessed whether the symptoms of the disease have been alleviated by any clinical testing method commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present invention (e.g., treatment methods or articles of manufacture) may not be effective in alleviating symptoms of each target disease, any statistical test method known in the art such as Student's t-test, chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of patients.

"Conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in proteins by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), This allows changes to be made frequently without altering the biological activity of the protein. Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. ., P. 224, (4th edition)). In addition, structurally or functionally similar amino acid substitutions are unlikely to disrupt biological activity.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dose of administration, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

"Exogenous" refers to a substance that is produced outside the organism, cell, or human as appropriate. "Endogenous" refers to a substance that is produced in a cell, organism, or human body as appropriate.

"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position of. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100. For example, when the sequences are optimally aligned, if 10 positions in the two sequences have 6 matches or homology, then the two sequences are 60% homologous; if 100 positions in the two sequences have 95 matches Or homology, then the two sequences are 95% homologous. In general, comparisons are made when the two sequences are aligned for the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of metastases. It should also be understood that due to intentional or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells. Where different names are meant, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a specific amount of nucleic acid, RNA, and / or DNA is amplified, as in, for example, US Patent No. 4,683,195. Generally speaking, it is necessary to obtain sequence information from the end of the target region or beyond so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5 'terminal nucleotides of the two primers may coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; edited by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample. The method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or produce A specific part of a nucleic acid.

"As needed" or "as needed" means that the event or environment described later can, but does not have to occur, and the description includes situations where the event or environment occurs or does not occur. For example, "comprising 1 to 3 antibody heavy chain variable regions as needed" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as a physiological / pharmaceutically acceptable carrier And excipients. The purpose of the pharmaceutical composition is to promote the administration to the living body, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.

In addition, the present invention relates to a method for immunodetection or determination of B7H3, a reagent for immunodetection or determination of B7H3, a method for immunodetection or determination of B7H3 expressing cells, and diagnosis for diagnosis of diseases related to B7H3 positive cells An agent comprising, as an active ingredient, a monoclonal antibody or an antibody fragment that specifically recognizes human B7H3 of the present invention and binds to the amino acid sequence of the extracellular region or its three-dimensional structure.

In the present invention, the method for detecting or determining the amount of B7H3 may be any known method. For example, it includes immunological detection or assay methods.

The immunodetection or measurement method is a method of detecting or measuring the amount of antibody or antigen using a labeled antigen or antibody. Examples of the immunodetection or measurement method include radioactive substance-labeled immune antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, western blot, physical chemistry Method, etc.

The above-mentioned diseases related to B7H3-positive cells can be diagnosed by detecting or measuring B7H3-expressing cells with the monoclonal antibody or antibody fragment of the present invention.

In order to detect a cell expressing a polypeptide, a known immunodetection method can be used, and an immunoprecipitation method, a fluorescent cell staining method, an immune tissue staining method, or the like is preferably used. In addition, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present invention, there is no particular limitation on a living sample for detecting or measuring B7H3, as long as it has the possibility of including cells expressing B7H3, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, Tissue fluid or culture fluid.

The diagnostic agent containing the monoclonal antibody or the antibody fragment thereof of the present invention may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction according to a required diagnostic method. Reagents for performing an antigen-antibody reaction include buffers, salts, and the like. The reagent for detection includes reagents commonly used in immunodetection or measurement methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, and a matrix corresponding to the label.

The term "cytotoxic drug" refers to a chemical molecule in a tumor cell that strongly disrupts its normal growth. Cytotoxic drugs can, in principle, kill tumor cells at a sufficiently high concentration, but due to the lack of specificity, while killing tumor cells, it will also cause apoptosis in normal cells, leading to serious side effects. The term is intended to include radioisotopes (such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ^32, and Lu radioactive isotopes), chemotherapeutics, toxins such as bacteria, fungi Small toxins or enzymatically active toxins of plant, animal or animal origin, including fragments and / or variants thereof.

The term "toxin" refers to small-molecule toxins and their derivatives from bacteria, fungi, plants or animals, including maytansinoids and their derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO2016 / 127790A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, acacia Arin chain, 蒴Lotus root toxin (modeccin), α-sarcin, sarcin, Aleutitesfordii toxin protein, dianthin toxin protein, Phytolaca americana toxin protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin aspergillus constrictor Restrictocin, phenomycin, economycin, and trichothecenes.

MMAF and MMAE are auristatin derivatives, see US2005 / 0238649 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. The structural formula is as follows:

Specifically, the auristatin / dorastatin drug modules such as MMAF and its derivatives can be prepared using methods described in US2005-0238649A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124. Oristatin / dolastatin pharmaceutical modules such as MMAE and its derivatives can be prepared using the method described in Doronina et al. (2003) Nat. Biotech. 21: 778-784. Drug-linker modules MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE can be conveniently synthesized by general methods, such as Doronina et al. (2003) Nat. Biotech. 21: 778- 784 and US Patent Application Publication No. US2005 / 0238649A1, and then conjugate them to the antibody of interest.

The drug 2852 described in the present invention, see WO2016 / 127790A1 (Compound Example 7), its structural formula is as follows:

By the method described in the present invention (Compound Example 8) or a method that can be inferred by those skilled in the art, the structural formula of the drug-linker module (3024 described in the present invention) is as follows:

The term "chemotherapeutic agent" is a chemical compound used in the treatment of tumors. Examples of chemotherapeutic drugs include alkylating agents such as thiotepa; cyclosphamide (CYTOXAN ^™ ); alkyl sulfonates such as busulfan, improsulfan, and tritium Piposulfan; aziridine such as benaodopa, carboquone, meturedopa and uredopa; aziridine and formazan Methylmelamine includes altretamine, triethylenemelamine, triethylenephosphoramidine, triethylenephosphorylamine and trimethylolomelamine; nitrogen mustard nitrogenmustards) such as phenyl butyrate nitrogen mustard, naphthalene mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxamphetamine hydrochloride; levamphetamine Acid nitrogen mustard (melphalan), new nitrogen mustard (novembichin), cholesterol phenylacetic acid nitrogen mustard, prednimustine, trofosfamide, uracil nitrogen mustard; nitrosureas such as Nitrourea, carmustine, chlorozotocin, fotemust ine), lomustine, nimustine, ranimustine; antibiotics such as aclamycin, actinomycin, autramycin, diazoserine, bleomycin , Actinomycin C (cactinomycin), calicheamicin, carabicin, chromomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin , Detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idabe Idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin ), Puromycin, quelamycin, rodorubicin, streptozotocin; streptozocin, tuberculin, ubenimex, net Statin (zinostatin), zorubicin; antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as dimethyl folic acid (den (opterin), methotrexate, tetrodine, trimetrexate; fludarabine, 6-mercaptopyridine, thiomiopterin, thioguanopterin; pyrimidine analogs Such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxitl uridine, and Enocitabine, fluorouridine, 5-FU; androgens such as calusterone, drostmostanolong propionate, epitiostanol, mepitiostane , Testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as aldehyde folic acid (frolinic acid); acetolactone Aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; biasntrene; edatraxate ); Defofamine; colchicine; diaziquone; eflomithine Elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; Mopidamol; nitroracrine; pintostatin; phenamet; pirarubicin; podophyllinic acid; 2- Ethyl hydrazine; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; finely streptozoic acid; triimine quinone; 2, 2 ', 2 "-trichlorrotriethylamine;urethan;vinblastamine;dacarbazine; mannitol nitrogen mustard; dibromonitol; mibrronitol Alcohols; pipobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa; taxanes, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); benzene Nitrogen butyrate; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinca tincture; platinum; etoposide (VP- 16); Ifosphaline; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide (teniposide); daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO ); Retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. This definition also includes antihormonal agents that can modulate or inhibit the effects of hormones on tumors, such as antiestrogens including tamoxifen, raloxifene, aromatase inhibitor 4 (5) -imidazole , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and fareston; and antiandrogenic agents such as fluorine Flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and a pharmaceutically acceptable salt of any of the above, an acid or derivative.

The term "linker unit" in the present invention is L1 and L2, and refers to a chemical structure fragment or bond covalently connected to an antibody at one end and a cytotoxic drug at the other end.

The term "drug loading" refers to the average number of cytotoxic drugs loaded on each ligand in the molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be per ligand (Pc) linkage 1 to 8 cytotoxic drugs. In the embodiment of the present invention, the drug loading amount is expressed as y, and the general method such as UV / visible light spectroscopy, mass spectrometry, ELISA test, and HPLC characteristics can be used to identify each ADC molecule after the coupling reaction. The average number of drugs.

In the present invention, y may be limited by the number of connected sites. In one embodiment of the present invention, the cytotoxic drug is coupled to the N-terminal amine group of the ligand and / or the ε-amino group of the lysine residue by a linker unit. Generally, the cytotoxic drug can react with the antibody during the coupling reaction. The number of conjugated drug molecules will be less than the theoretical maximum.

The following non-limiting methods can be used to regulate the load of antibody cytotoxic drug conjugates, including: (1) regulating the mole ratio of the linking reagent and the monoclonal antibody, (2) regulating the reaction time and temperature, (3) choosing a different Reaction reagent.

The term "carrier" used in the drug of the present invention refers to a system that can change the way the drug enters the human body and its distribution in the body, regulate the release rate of the drug, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and increase bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structure to form various forms of aggregates. Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.

The term "excipient" is an additive in a pharmaceutical formulation other than the main drug and may also be referred to as an excipient. Such as adhesives, fillers, disintegrating agents, lubricants in tablets; matrix parts in semi-solid preparations ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, co-solvents in liquid preparations , Emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. can all be called excipients.

The term "diluent" is also called a bulking agent, and its main purpose is to increase the weight and volume of a tablet. The addition of the diluent not only ensures a certain volume size, but also reduces the dose deviation of the main components and improves the compression moldability of the drug. When the medicine of the tablet contains an oily component, it is necessary to add an absorbent to absorb the oily substance, so as to keep the "dry" state, so as to facilitate the preparation of the tablet.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. The injection or microemulsion can be injected into the patient's bloodstream by local injection. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of a compound of the invention. To maintain this constant concentration, continuous intravenous drug delivery devices can be used. An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.

The pharmaceutical composition may be in the form of a sterile injectable water or oily suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent. In addition, a sterile fixed oil can be conveniently used as a solvent or suspension medium.

The invention also relates to a method for treating diseases associated with human B7H3-positive cells, in particular the use in the treatment of cancer and inflammation.

2. Examples and test examples

The present invention is further described below with reference to examples, but these examples and test examples do not limit the scope of the present invention. The experimental methods without specific conditions in the examples or test examples of the present invention generally follow general conditions, such as the manual of antibody technology experiments and molecular breeding manuals of Cold Spring Harbor; or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific source are general reagents purchased on the market.

Example 1. Preparation of B7H3 antigen and detection protein

The human B7H3 shown in SEQ ID NO: 1 is used as a template of the B7H3 of the present invention to design the amino acid sequence of the antigen and the protein for detection of the present invention. The following B7H3 antigens refer to human B7H3 unless otherwise specified.

1.1 Human B7H3 full-length amino acid sequence: B7H3 (SEQ ID NO: 1): Note: The double bottom line is the signal peptide (Signal peptide: 1-28); the bottom line is the B7H3 extracellular domain (29-466), of which 29-139 are Ig-like V-type 1 domains, 145-238 are Ig-like C2-type 1 domains; 243-357 are Ig-like V-type 2 domains; 363-456 are Ig-like C2-type 2 domains; the dashed-dotted line is the transmembrane region Part (Transmembrane domain: 467-487); part in italics (Cytoplasmic domain: 488-534).

1.2 Full-length amino acid sequence of murine B7H3 (SEQ ID NO: 2) Note: The double bottom line is the signal peptide (Signal peptide: 1-28); the bottom line is the B7H3 extracellular domain (29-248), of which 29-139 are Ig-like V-type domains, 145 -238 is an Ig-like C2-type domain; the dotted line portion is a transmembrane domain portion (Transmembrane domain: 249-269); the italic portion is an intracellular region (Cytoplasmic domain: 270-316).

1.3 Screening and detection of human B7H3 antigen (SEQ ID NO: 3)

For commercial products (R & D cat # 1949-B3-050 / CF, referred to as 2Ig-B7H3), the sequence is as follows: Note: The underlined part is the extracellular region of B7H3; the italics are His-tag.

1.4 Detection of human B7H3 antigen (SEQ ID NO: 4)

It is a commercial product (Sino Biological cat # 11188-H08H, referred to as 4Ig-B7H3), the sequence is as follows: Note: The underlined part is the extracellular region of B7H3; the italics are His-tag.

1.5 Screening and detection of murine B7H3 antigen (SEQ ID NO: 5)

For commercial products (R & D cat # 1397-B3-050 / CF), the sequence is as follows: Note: The underlined part is the extracellular region of B7H3; the italics are His-tag.

Example 2. Preparation of fully human antibodies

2.1 Screening of positive sequences

Human PBMC, spleen, and lymph node tissues were used to isolate B cells, and RNA was extracted to construct a natural single-chain phage antibody library (storage capacity 3.2 × 10 ¹⁰ ). The constructed single-stranded phage library was packaged to form phage particles, and then sieved by liquid phase method. The phage was combined with biotinylated B7H3 in liquid phase, and then separated by streptavidin magnetic beads. In order to obtain positive sequences that can be cross-bound to human B7H3 (R & D cat # 1949-B3-050 / CF) and murine B7H3 (R & D cat # 1397-B3-050 / CF), respectively, biotinylated human B7H3 and biological Primed rat B7H3 was screened alternately. The first round was screened with 2 μg / ml biotinylated human B7H3. The second round was screened with 2 μg / ml biotinylated rat B7H3. The third round was screened with 0.5 μg / ml. Biotinylated human B7H3 was screened. After three rounds of screening, 500 simple strains were picked and packaged into phages for phage ELISA testing. The binding activity of the simple strain phage to human B7H3 (R & D cat # 1949-B3-050 / CF) and mouse B7H3 (R & D cat # 1397-B3-050 / CF) was tested respectively: 1 μg / ml human B7H3 was coated on the ELISA plate Or murine B7H3 and 1% BSA, add 1: 1 phage supernatant diluted in blocking buffer, and finally detect with anti-M13HRP; Divide the OD450 value measured by ELISA greater than 0.5, and the ELISA OD450 value of human and murine B7H3 divided by Pure strains combining two ratios of ELISA OD450 values of 1% BSA greater than 2.0 were sequenced to obtain 9 specific sequences.

2.2 Construction of a complete monoclonal antibody

Nine specific sequences obtained from phage library screening were used to construct intact antibodies. ELISA binding experiments were performed to determine that two of these antibodies had strong binding, h1702 and h1703, respectively. The process of constructing its entire monoclonal antibody is as follows: According to the sequence of the single-chain antibody obtained by sequencing, primers PCR are designed to construct the VH / VK / VL gene fragments of each single-chain antibody sequence. The heavy and light chain variable regions of h1702 and h1703 were obtained.

> h1702 heavy chain variable region sequence SEQ ID NO: 6

> h1702 light chain variable region sequence SEQ ID NO: 7

> h1703 heavy chain variable region sequence SEQ ID NO: 8

> h1703 light chain variable region sequence SEQ ID NO: 9

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italics in the sequence are the FR sequence, and the bottom line is the CDR sequence.

The CDR sequences in the light and heavy chains of each antibody are shown in Table 1.

The antibody variable region was homologously recombined with the constant region gene (CH1-FC / CL) fragment to construct a complete antibody VH-CH1-FC / VK-CL / VL-CL.

The complete full-length antibody h1702 and h1703 were constructed as follows: h1702: h1702 heavy chain (IgG1) amino acid sequence: (SEQ ID NO: 22)

h1702 light chain amino acid sequence: Lamada (SEQ ID NO: 23)

h1703: h1703 heavy chain (IgG1) amino acid sequence: (SEQ ID NO: 24)

h1703 light chain amino acid sequence: Kappa (SEQ ID NO: 25)

In order to further improve the stability of the antibody, amino acid mutation was performed on the light chain sequence of h1702, specifically the first amino acid residue Q at the N-terminus of the light chain (SEQ ID NO: 23) was replaced by D, and the mutation C was deleted. The first amino acid residue S is terminated to obtain a more stable and uniform monoclonal antibody h1702-DS.

The heavy chain sequence of h1702-DS after mutation modification is SEQ ID NO: 22, and the light chain amino acid sequence is as follows: (SEQ ID NO: 26).

2.3 Expression and purification of fully human antibodies

The plastids expressing the light and heavy chains of the antibodies were transfected into HEK 293E cells at a ratio of 1.5: 1. After 6 days, the supernatant was collected, and the impurities were removed by high-speed centrifugation. Flush the column with PBS until the A280 reading drops to baseline. The target protein is extracted with an acidic extraction solution of pH 3.0-pH 3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. The extracted sample was appropriately concentrated, and further purified using Superdex 200 (GE) gel chromatography balanced with PBS to remove the polymer and collect the monomer peaks for use in equipment.

B7H3 antibody-drug conjugate preparation example

Example 3 Preparation of Murine Antibody

3.1 Preparation of Antigen

The human B7-H3 (h-B7H3-Fc) sequence encoding the huFc tag was synthesized by Integrated DNA Technology (IDT) (the above B7-H3 recombinant proteins were designed by the template sequence of the present invention), and were selected and cloned into pTT5 vectors (Biovector ). After the recombinant B7-H3 protein is expressed in 293T cells, it is purified by general techniques in the art. The purified protein can be used for mouse immunization to obtain antibodies.

h-B7H3-Fc sequence: SEQ ID NO: 27

3.2 Preparation of antibodies

Anti-human B7H3 monoclonal antibodies were produced by immunizing mice. Swiss Webster white mice, female, 6 weeks old (Charles River) were used in the experiments. Rearing environment: SPF level. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12 / 12-hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. The immune antigen was an Fc-tagged human B7H3 recombinant protein (huB7H3-Fc). Titermax (sigma Lot Num: T2684) was used as an adjuvant. The ratio of the antigen to the adjuvant (titermax) is 1: 1. The antigen is emulsified and vaccinated for the first 0, 21, 35, 49, 63 days. On day 0, 15 μg + 25 / foot of emulsified antigen were injected intraperitoneally (IP). On days 21, 35, 49, and 63, intraperitoneal (IP) injection of 15 μg + footpad (post-emulsified antigen) 15 / head, to strengthen immunity 3 days before splenocyte fusion, intraperitoneal (IP) injection of 15 μg + footpad (footpad) ) 15 / antigen solution in normal saline. Blood tests were performed on days 42, 56, and 70, and mouse sera were detected by ELISA and FACS methods to determine antibody titers in mouse sera. After the 5th immunization, mice with high antibody titers and plateaus in the serum were selected for splenocyte fusion, and spleen lymphocytes were splenocytes and myeloma cells Sp2 / 0 cells (ATCC® CRL) using an optimized electrofusion procedure. -8287 ^(TM )) to obtain fusion tumor cells.

After the fusion of the fused tumor cells for 7-14 days, the supernatant of the culture medium was taken, and the B7-H3 recombinant protein was used for antibody screening of the fused tumor supernatant by an ELISA experiment. The positive antibody strain obtained was further used to stably express B7-H3. CHO-S cells were compared with blank CHO-S cells to exclude non-specifically bound antibody fusion tumor strains, and screened by flow sorting to select two fusion tumors that bound recombinant proteins and also expressed antigens on the cells. Fusion tumor cells in logarithmic growth phase were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (Prime Script ^™ Reverse Transcriptase, Takara # 2680A). The reverse-transcribed cDNA was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B0503) and then sequenced to obtain the sequence of the mouse-derived antibody m1704.

The sequence of the variable region of the heavy and light chains of the mouse monoclonal antibody m1704 is as follows: m1704 heavy chain SEQ ID NO: 28

m1704 light chain SEQ ID NO: 29

In order to improve the stability of the antibody, the CDR sequences of the above-mentioned murine antibodies were optimized. The optimized CDR region has the following sequence:

The heavy and light chain variable regions of the mouse antibody were cloned into pTT vector plastids (Biovector) containing human IgG1 heavy chain constant region and kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells. The chimeric antibody of B7-H3 can be used after purification according to general technical methods.

3.3 Humanization of mouse antibodies

Humanization of the murine anti-human B7-H3 monoclonal antibody was performed as described in many literatures in the art. In short, the human constant domain is used instead of the parent (murine-derived antibody) constant domain, and the human antibody sequence is selected based on the homology of the mouse-derived antibody and the human antibody. The present invention humanizes the antibody m1704.

Based on the obtained typical structure of the mouse-derived antibody VH / VLCDR, the heavy and light chain variable region sequences were compared with the human-derived antibody germline database to obtain a human germline template with high homology. The human germline light chain framework region is derived from the human kappa light chain gene human germline light chain template IGkV1-33, and the human germline heavy chain framework region is from the human heavy chain template IGHV3-23.

The CDR region of mouse-derived antibody m1704 was transplanted onto the selected corresponding humanized template, and the humanized variable region was replaced, and then recombined with the IgG constant region (preferably the heavy chain is IgG1 and the light chain is κ). Then, based on the three-dimensional structure of the mouse-derived antibody, back-mutate the embedded residues, the residues that directly interact with the CDR regions, and the residues that have an important influence on the conformation of VL and VH, and CDR regions Chemically labile amino acid residues were optimized. Antibodies composed of the following humanized light and heavy chain variable region sequences were designed and tested.

h1704VH1 (SEQ ID NO: 36):

h1704VH2 (SEQ ID NO: 37):

h1704VL1 (SEQ ID NO: 38):

h1704VL2 (SEQ ID NO: 39):

After comparing the number of expression tests and back mutations, the final humanized h1704-3 antibody molecule (using the VH1 heavy chain variable region and the VL2 light chain variable region) was selected, and its heavy and light chain sequences are as SEQ ID NO: 40 and 41. h1704-3 antibody heavy chain (IgG1) sequence: SEQ ID NO: 40

h1704-3 antibody light chain (Kappa) sequence: SEQ ID NO: 41

A cDNA fragment was synthesized based on the gene sequences of the light and heavy chains of each humanized antibody, and inserted into a pcDNA 3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1: 2, and incubated in a CO ₂ incubator for 4-5 day. The expressed antibody is recovered by centrifugation, and the antibody is purified to obtain the humanized antibody protein of the present invention.

B7H3 antibody-drug conjugate preparation example

The following Examples 4 to 11 are preparation processes of ADCs related to the present invention. The antibody (h1702, h1703) in Example 4 to Example 7 was prepared by coupling a drug (MMAF or 3024) with a linking unit to its lysine acid to prepare an ADC; Examples 8 to 11 were prepared by The thiol group on the cysteine of the antibody (h1702, h1704-3, h1702DS) is reacted with a drug (3024) with a linking unit to prepare ADC.

Example 4: Preparation of B7H3-h1702-L2-MC-MMAF (h1702-MMAF)

Dissolve S- (3-carbonylpropyl) thioacetate (0.10 mg, 0.75 μmol) in 0.2 mL of acetonitrile solution and set aside; add B7H3-h1702, pH = 4.3 in acetic acid / sodium acetate buffer (2.5 mg) / ml, 2.0mL, 0.034umol) was added to the above-prepared acetonitrile solution of S- (3-carbonylpropyl) thioacetate, and then 0.1mL of an aqueous solution of sodium cyanoborohydride (3.4mg, 53μmol) was added dropwise, The reaction was shaken at 25 ° C for 2 hours. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent phase: 0.05M PBS buffer solution with a pH of 6.5, containing 0.001M EDTA), and the unreacted thioacetic acid S- (3-carbonyl group was removed. Propyl) ester and sodium cyanoborohydride to obtain the title product 1b in a PBS buffer solution (about 5.5 mL), and concentrated by ultrafiltration to about 2.5 ml for further reaction.

Second step

To 1b of the PBS buffer solution (2.5mL) was added 0.07mL of a 2.0M hydroxylamine hydrochloride solution. After the addition was completed, it was placed in a water bath shaker and the reaction was shaken at 25 ° C for 30 minutes to stop the reaction. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent: 0.05M PBS buffer solution with pH 6.5 and 0.001M EDTA) to obtain the title product B7H3-h1702 MAb -propanethiol 1c PBS buffer solution (concentration 0.84 mg / ml, 5.0 mL).

third step

Compound (S) -2-((2R, 3R) -3-((1S, 3S, 5S) -2-((3R, 4S, 5S) -4-((S) -2-((S) -2- (6- (2,5-dicarbonyl-2,5-dihydro-1H-pyrrole-1-yl) -N-methylhexylamine) -3-methylbutanidine) -N, 3-dimethylbutanylamine) -3-methoxy-5-methylheptanyl) -2-azabicyclo [3.1.0] hexane-3-yl) -3-methoxy-2 -Methylpropionamine) -3-phenylpropionic acid MC-MMAF (0.32mg, 0.34μmol) was dissolved in 0.5mL acetonitrile, and B7H3-h1702 monoclonal antibody -propanethiol PBS buffer solution 1c (0.84mg / mL, 5.0 mL), put in a water bath shaker, and shake the reaction at 25 ° C for 4 hours to stop the reaction.

The reaction solution was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution with pH 6.5 and 0.001M EDTA) to obtain the title product h1702-MMAF in PBS buffer (0.21mg / mL , 14.5 mL) and stored frozen at 4 ° C. Q-TOFLC / MS calculation average: y = 1.76.

Example 5: Preparation of B7H3-h1703-L2-MC-MMAF (h1703-MMAF)

Dissolve S- (3-carbonylpropyl) thioacetate (0.11mg, 0.82μmol) in 0.2mL acetonitrile solution, set aside; to antibody B7H3-h1703, pH = 4.3 in acetic acid / sodium acetate buffer (2.5mg / ml, 2.0mL, 0.034umol) was added to the above-prepared acetonitrile solution of S- (3-carbonylpropyl) thiothioacetate, and then 0.1mL of an aqueous solution of sodium cyanoborohydride (3.4mg, 53μmol) was added dropwise, The reaction was shaken at 25 ° C for 2 hours. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent phase: 0.05M PBS buffer solution with a pH of 6.5, containing 0.001M EDTA), and the unreacted thioacetic acid S- (3-carbonyl group was removed. Propyl) ester and sodium cyanoborohydride to obtain the title product 2b in a PBS buffer solution (about 5.5 mL), and concentrated by ultrafiltration to about 2.5 ml for further reaction.

Second step

To 2b of the PBS buffer solution (2.5 mL) was added 0.07 mL of a 2.0 M hydroxylamine hydrochloride solution. After the addition was completed, it was placed in a water bath shaker, and the reaction was shaken at 25 ° C for 30 minutes to stop the reaction. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent: 0.05M PBS buffer solution with pH 6.5, containing 0.001M EDTA) to obtain the title product B7H3-h1703 mAb -propanethiol 2c PBS buffer solution (concentration 0.82 mg / ml, 5.0 mL).

third step

Compound ( S ) -2-(( 2R , 3R ) -3-(( 1S , 3S , 5S ) -2-(( 3R , 4S , 5S ) -4-(( S ) -2-(( S ) -2- (6- (2,5-dicarbonyl-2,5-dihydro-1 H -pyrrole-1-yl) -N -methylhexylamine) -3- Methylbutyramine) -N , 3-dimethylbutyramine) -3-methoxy-5-methylheptyl) -2-azabicyclo [3.1.0] hexane-3-yl ) -3-methoxy-2-methylpropanamine) 3-phenylpropanoic acid MC-MMAF (0.32mg, 0.34μmol) was dissolved in 0.5mL of acetonitrile, and B7H3-h1703 monoclonal antibody -propanethiol PBS was added The buffer solution 2c (0.82 mg / mL, 5.0 mL) was placed in a water bath shaker, and the reaction was stopped after shaking for 4 hours at 25 ° C.

The reaction solution was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution with pH 6.5, containing 0.001M EDTA) to obtain the title product h1703-MMAF in PBS buffer (0.20mg / mL , 13.0 mL), and stored frozen at 4 ° C.

Q-TOFLC / MS calculation average: y = 2.29.

Example 6: Preparation of B7H3-h1702-L2-3024 (h1702-3024)

Dissolve S- (3-carbonylpropyl) thioacetate (0.45mg, 3.38μmol) in 0.8mL acetonitrile solution, set aside; add antibody B7H3-h1702, pH = 4.3 in acetic acid / sodium acetate buffer (5.0mg / ml, 8.0 mL, 0.270 μmol) was added to the above-prepared acetonitrile solution of S- (3-carbonylpropyl) thioacetate, and then 0.1 mL of an aqueous solution of sodium cyanoborohydride (12.8 mg, 0.2 mmol) was added dropwise. The reaction was shaken at 25 ° C for 2 hours. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent phase: 0.05M PBS buffer solution with a pH of 6.5, containing 0.001M EDTA), and the unreacted thioacetic acid S- (3-carbonyl group was removed. Propyl) ester and sodium cyanoborohydride to give the title product 3b in a PBS buffer solution (about 14.5 mL), and concentrated by ultrafiltration to about 7.5 ml for the next reaction.

Second step

To 3b of the PBS buffer solution (7.5 mL) was added 0.20 mL of a 2.0 M hydroxylamine hydrochloride solution. After the addition was completed, the solution was placed in a water bath shaker, and the reaction was shaken at 25 ° C for 30 minutes to stop the reaction. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent: 0.05M PBS buffer solution with pH 6.5, containing 0.001M EDTA) to obtain the title product B7H3-h1702 mAb -propanethiol 3c PBS buffer solution (concentration 2.84 mg / ml, 14.0 mL).

third step

Compound ( S ) -2-(( 2R , 3R ) -3-(( 1S , 3S , 5S ) -2-(( 3R , 4S , 5S ) -4-(( S ) -2-(( S ) -2- (6- (2,5-dicarbonyl-2,5-dihydro-1 H -pyrrole-1-yl) -N -methylhexylamine) -3- Methylbutanidine) -N, 3-dimethylbutanamine) -3-methoxy-5-methylheptanyl) -2-azabicyclo [3.1.0] hexane-3-yl ) -3-methoxy-2-methylpropanamine) -3- (2-fluorophenyl) propionic acid compound 3024 (2.6 mg, 2.7 μmol) was dissolved in 1.4 mL of acetonitrile, and B7H3-h1702 monoclonal antibody was added -Propanethiol PBS buffer solution 3c (2.84 mg / mL, 14.0 mL), placed in a water bath shaker, and shaken at 25 ° C for 4 hours to stop the reaction.

The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent: 0.05M PBS buffer solution with pH 6.5 and 0.001M EDTA) to obtain the title product h1702-3024 in PBS buffer (1.35mg / mL , 27.5 mL), and stored frozen at 4 ° C.

Q-TOFLC / MS calculation average: y = 2.05.

Example 7: Preparation of B7H3-h1703-L2-3024 (h1703-3024)

Dissolve S- (3-carbonylpropyl) thioacetate (0.50mg, 3.78 μmol) in 0.8mL acetonitrile solution and set aside; to antibody B7H3-h1703, pH = 4.3 in acetic acid / sodium acetate buffer (5.0mg / ml, 8.0 mL, 0.270 umol) was added to the above-prepared acetonitrile solution of S- (3-carbonylpropyl) thioacetate, and then 0.1 mL of an aqueous solution of sodium cyanoborohydride (12.8 mg, 0.2 mmol) was added dropwise. The reaction was shaken at 25 ° C for 2 hours. The reaction solution was purified by desalting with a Sephadex G25 gel column (eluent phase: 0.05M PBS buffer solution with a pH of 6.5, containing 0.001M EDTA), and the unreacted thioacetic acid S- (3-carbonyl group was removed. Propyl) ester and sodium cyanoborohydride to obtain the title product 4b in a PBS buffer solution (about 14.0 mL), and concentrated by ultrafiltration to about 7.5 ml for the next reaction.

Second step

To 4b of PBS buffer solution (7.5 mL) was added 0.20 mL of a 2.0 M hydroxylamine hydrochloride solution. After the addition was completed, the solution was placed in a water bath shaker and the reaction was shaken at 25 ° C for 30 minutes to stop the reaction. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution with pH 6.5, containing 0.001M EDTA) to obtain the title product B7H3-h1703 mAb -propanethiol 4c PBS buffer solution (concentration 2.64 mg / ml, 14.5 mL).

third step

Compound ( S ) -2-(( 2R , 3R ) -3-(( 1S , 3S , 5S ) -2-(( 3R , 4S , 5S ) -4-(( S ) -2-(( S ) -2- (6- (2,5-dicarbonyl-2,5-dihydro-1 H -pyrrole-1-yl) -N -methylhexylamine) -3- Methylbutyramine) -N , 3-dimethylbutyramine) -3-methoxy-5-methylheptyl) -2-azabicyclo [3.1.0] hexane-3-yl ) -3-methoxy-2-methylpropanamine) -3- (2-fluorophenyl) propionic acid compound 3024 (2.6 mg, 2.7 μmol) was dissolved in 1.45 mL of acetonitrile, and B7H3-h1703 monoclonal antibody was added -Propanethiol PBS buffer solution 6c (2.64 mg / mL, 14.5 mL), put in a water bath shaker, and shake the reaction at 25 ° C for 4 hours to stop the reaction.

The reaction solution was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution with pH 6.5 and 0.001M EDTA) to obtain the title product h1703-3024 in PBS buffer (1.20mg / mL , 28.0 mL), and stored frozen at 4 ° C.

Q-TOFLC / MS calculation average: y = 1.62.

Example 8. Preparation of B7H3-h1702-cys-3024 (h1702-cys-3024)

Add 0.01 mM tris (2-carboxyethyl) phosphine (TCEP) to the antibody h1702, pH = 6.5 in 0.05 M PBS buffer solution (10.0mg / ml, 5.0mL, 0.333umol) at 37 ° C. 0.07mL (0.7umol) of a 10 mM aqueous solution was placed in a water bath shaker, and the reaction was shaken at 37 ° C for 3 hours to stop the reaction; the reaction solution was cooled to 25 ° C with a water bath, and the compound ( S ) -2-(( 2 R , 3 R ) -3-((1 S , 3 S , 5 S ) -2-((3 R , 4 S , 5 S ) -4-(( S ) -2-(( S ) -2 -(6- (2,5-dicarbonyl-2,5-dihydro-1 H -pyrrole-1-yl) -N -methylhexamidine) -3-methylbutanidine) -N , 3 -Dimethylbutyramine) -3-methoxy-5-methylheptyl) -2-azabicyclo [3.1.0] hexane-3-yl) -3-methoxy-2- Methylpropanamine) -3- (2-fluorophenyl) propanoic acid SHR3024 (2.6 mg, 2.7 μmol) was dissolved in 0.5 mL of acetonitrile, added to antibody h1702 (containing TCEP) cooled to 25 ° C, and placed in Water bath shaker, shaking reaction at 25 ° C for 3 hours to stop the reaction; the reaction solution was desalted and purified using Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution with pH 6.5, containing 0.001 M EDTA) To obtain the title product in PBS buffer (6.85 mg / mL, 7.5 mL), frozen at 4 ° C Deposit.

HIC-HPLC calculation average: y = 3.5.

Example 9. Preparation of B7H3-h1704-3-cys-3024 (h1704-3-cys-3024)

Add 0.05 mM tris (2-carboxyethyl) to the antibody B7H3-h1704-3, pH = 6.5 in 0.05 M PBS buffer solution (10.0mg / ml, 5.0mL, 0.333umol) at 37 ° C. ) 0.075mL (0.75umol) of a 10 mM aqueous solution of phosphine (TCEP), put it in a water bath shaker, shake the reaction at 37 ° C for 3 hours, and stop the reaction; cool the reaction solution to 25 ° C with a water bath, and then compound ( S )- 2-((2 R , 3 R ) -3-((1 S , 3 S , 5 S ) -2-((3 R , 4 S , 5 S ) -4-(( S ) -2-(( S ) -2- (6- (2,5-dicarbonyl-2,5-dihydro-1 H -pyrrole-1-yl) -N -methylhexamidine) -3-methylbutanidine) -N , 3-dimethylbutanamine) -3-methoxy-5-methylheptyl) -2-azabicyclo [3.1.0] hexane-3-yl) -3-methoxy Methyl-2-methylpropanamine) -3- (2-fluorophenyl) propionic acid compound 3024 (2.6 mg, 2.7 μmol) was dissolved in 0.5 mL of acetonitrile, and added to the antibody B7H3-h1704- 3 (containing TCEP), placed in a water bath shaker, and shaken the reaction at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS at pH 6.5). Buffered aqueous solution containing 0.001 M EDTA) to obtain the title product in PBS buffer (6.75 mg / mL, 7 .8 mL) and stored frozen at 4 ° C.

HIC-HPLC calculated average: y = 3.4.

Example 10. Preparation of B7H3-h1702DS-cys-3024 (h1702DS-cys-3024)

Add 0.01mM tris (2-carboxyethyl) phosphine to the antibody H1702-DS, 0.05 M PBS buffer solution (10.0mg / ml, 2.0mL, 0.133umol) at pH 6.5 at 37 ° C. (TCEP) 0.028 mL (0.28umol) of 10 mM aqueous solution, put it in a water bath shaker, shake the reaction at 37 ° C for 3 hours, and stop the reaction; cool the reaction solution to 25 ° C with a water bath, and then compound ( S ) -2- ((2 R , 3 R ) -3-((1 S , 3 S , 5 S ) -2-((3 R , 4 S , 5 S ) -4-(( S ) -2-(( S ) -2- (6- (2,5-dicarbonyl-2,5-dihydro-1 H -pyrrole-1-yl) -N -methylhexamidine) -3-methylbutanidine) -N , 3-Dimethylbutanidine) -3-methoxy-5-methylheptyl) -2-azabicyclo [3.1.0] hexane-3-yl) -3-methoxy- 2-methylpropanamine) -3- (2-fluorophenyl) propionic acid compound 3024 (1.02 mg, 1.1 μmol) was dissolved in 0.2 mL of acetonitrile, and added to the antibody h1702-DS (containing TCEP) which had been cooled to 25 ° C. ), Place in a water bath shaker and shake the reaction at 25 ° C for 3 hours to stop the reaction. The reaction solution is desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to give the title product in PBS buffer (6.65 mg / mL, 2.7 mL) 4 ℃ stored frozen.

HIC-HPLC calculation average: y = 3.55.

The following test method is used to verify the performance and beneficial effects of the antibodies of the present invention.

Test example 1. ELISA binding experiment

In order to detect the B7H3 antibodies screened and the in vitro binding capacity of different B7H3-ADCs to different forms of B7H3 in humans, human 2Ig-B7H3 (Cat. # 1949-B3-050 / CF, R & D) and human 4Ig-B7H3 (Cat # 11188-H08H, SinoBiological) was used for in vitro binding assays.

Human B7H3 protein (2Ig / 4Ig) was diluted to a concentration of 1 μg / ml with PBS (Sigma, P4417-100TAB) buffer at pH 7.4, and added to a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 100 μl / well. , Left at 4 ° C for 16-20 hours overnight. After discarding the liquid, 120 μl / well of a 5% skim milk (bright skim milk) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added, and the mixture was blocked by incubation at 37 ° C. for 2 hours. After blocking, discard the blocking solution and wash the plate 4 times with PBST buffer. Then add 100 μl / well of the corresponding B7H3 antibody (or B7H3-ADC) with an initial concentration of 1 μM. Dilute 8 gradients with PBST buffer multiple times. Incubate for 1 hour in a 37 ° C incubator. After incubation, discard the reaction solution in the microtiter plate, wash the plate 4 times with PBST, and add 100 μl / well of HRP-labeled goat anti-human IgG (goat anti-human IgG) Fcγ fragment specific to PBST dilution (1: 4000). Secondary antibodies (Jackson Immuno Research, 109-005-008) were incubated at 37 ° C for 1 hour. After washing the plate 4 times with PBST, add 100 μl / well TMB color development matrix (KPL, 52-00-03), incubate at room temperature for 3-5min, add 100 μl / well 1MH ₂ SO _{4 to} stop the reaction, and use a NOVOStar microplate reader The absorption value was read at 450 nm, and the EC50 value of the antibody binding to the antigen was calculated. The results are shown in Table 2.

Conclusion: For human 2Ig-B7H3 and human 4Ig-B7H3, ADC and naked antibody have similar binding power in ELISA experiments, that is, the binding power was not reduced after ADC labeling.

Test example 2. Cross-bonding experiment with different species B7H3

In order to detect the screened B7H3 antibodies and the corresponding different B7H3-ADC, the in vitro binding capacity of B7H3 from different species, mouse B7H3 (Cat. # 1397-B3-050 / CF, R & D) was used to perform in vitro Combined detection.

The B7H3 protein (mouseB7H3) of different species was diluted to a concentration of 1 μg / ml with PBS (Sigma, P4417-100TAB) buffer at pH 7.4, and added to a 96-well microtiter plate at a volume of 100 μl / well, and left overnight at 4 ° C. 16-20 hours. After discarding the liquid, 120 μl / well of a 5% skim milk (bright skim milk) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added, and the mixture was blocked by incubation at 37 ° C. for 2 hours. After blocking, discard the blocking solution and wash the plate 4 times with PBST buffer. Then add 100 μl / well of the corresponding B7H3 antibody (or B7H3-ADC) with an initial concentration of 1 μM. Dilute 8 gradients with PBST buffer multiple times. Incubate for 1 hour in a 37 ° C incubator. After incubation, discard the reaction solution in the microtiter plate, wash the plate 4 times with PBST, add 100 μl / well of HRP-labeled goat anti-human IgG diluted with PBST (1: 4000), and a secondary antibody specific to the Fcγ fragment ( Jackson Immuno Research, 109-005-008), and incubated at 37 ° C for 1 hour. After washing the plate 4 times with PBST, add 100 μl / well TMB color development matrix (KPL, 52-00-03), incubate at room temperature for 3-5min, add 100 μl / well 1MH ₂ SO _{4 to} stop the reaction, and use a NOVOStar microplate reader The absorbance was read at 450 nm and the EC50 value of the antibody's binding to the antigen was calculated (see Table 3 for the results).

The results showed that h1702 and h1703, and different ADCs had relatively weak binding to murine B7H3, showing that the two monoclonal antibodies had good human B7H3 binding specificity.

Test example 3. Biacore antibody affinity test

Biacore and GE instruments were used to measure the affinity of anti-B7H3 antibodies, anti-B7H3-ADC and various antigens of human 2Ig-B7H3 antigen, human 4Ig-B7H3 antigen.

A biosensing chip ProteinA (Cat. # 29127556, GE) was used to affinity capture a certain amount of antibody / ADC to be tested, and then the human 2Ig-B7H3 antigen (Cat. # 1949- B3-050 / CF, R & D), human 4Ig-B7H3 antigen (Cat. # 11188-H08H, SinoBiological), real-time detection of reaction signals using a Biacore instrument (Biacore T200, GE) to obtain binding and dissociation curves. After the dissociation of each cycle was completed, the biochip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5) (Cat. # BR-1003-54, GE). The buffer used in the experiment was HBS-EP buffer solution (pH 7.4) (Cat. # BR-1001-88, GE).

The experimental data was fitted with BIA evaluation version 4.1, and GE software was fitted with the (1: 1) Langmuir model to obtain the affinity value. The experimental results are shown in Table 4.

Conclusion: Affinity tests on human 2Ig-B7H3 and human 4Ig-B7H3 in Biacore experiments showed that the affinity of ADC and naked antibody was similar.

Test example 4. In vitro cell binding experiment

In this experiment, the fluorescence signal of the antibody on the cell surface is detected, and the binding of the antibody is evaluated according to the intensity of the fluorescence signal. After 10 μg of primary antibody was incubated with 2 × 10 ⁵ U87MG cells on ice for 30 minutes, excess antibodies were washed away. Cells were incubated with APC anti-human IgG Fc (Biolegend, 409306) for 30 minutes at room temperature. After the excess antibodies were washed away, the fluorescence signal on the cell surface was read using BDVerse (see Figure 1 for results).

The results show that h1702 has strong binding ability to the B7H3 antigen on the cell surface.

Test example 5. In vitro cell endocytosis experiment

In this experiment, the intracellular antibody or different ADCs were used to detect the fluorescence signal, and the endocytosis effect of the antibody was evaluated according to the intensity of the fluorescence signal. B7H3 antibody / ADC and APC anti-human IgGFc (Biolegend, 409306) were mixed at a molar ratio of 1: 2 and incubated on ice for 15 minutes. After incubating the antibody mixture with 2 × 10 ⁵ U87MG cells for 30 minutes on ice, the excess antibodies were washed off, then the cells were transferred to a medium pre-warmed at 37 ° C, and incubated at 37 ° C for 0, 15, 30, 60 and 120 minutes. Centrifuge the cells and resuspend the cells in the antibody extract and incubate at room temperature for 7 minutes. Wash off the antibody extract and use BDVerse to read the intracellular fluorescence signal. See Figure 2, Figure 3, and Figure 4. The results showed that the endocytosis effect of ADC on U87MG cells was comparable to that of naked antibody.

Test example 6. Evaluation of SD rat T1 / 2

Four SD rats, half male and half male, 12/12 hours light / dark adjustment, temperature 24 ± 3 ℃ constant temperature, humidity 50-60%, free to eat and drink. Purchased from Jiejiejie Experimental Animal Co., Ltd. On the day of the experiment, SD rats were injected with the test drug B7H3 antibody / ADC in the tail vein at a dose of 3 mg / kg and an injection volume of 5 ml / kg.

Blood collection time points were: 5 minutes, 8 hours, and 24 hours (day 2), 3 days, 5 days, 8 days, 11 days, and 15 days after administration on day 1 200 μL each time (equivalent to taking 100 μL of serum); the collected blood samples were left at room temperature for half an hour to agglutinate, and then centrifuged at 10,000 × g for 10 minutes at 4 ° C. The supernatant was collected and immediately stored at -80 ° C.

The serum B7H3 antibody concentration was detected by ELISA, and the PK analysis was performed. The results are shown in Table 5.

The results show that the half-life of h1702 of the present invention in rats is about 185 hours (7.7 days).

The serum B7H3 antibody ADC concentration was detected by ELISA, and the PK analysis was performed. The results are shown in Table 6.

The results show that after intravenously administering 3mg / kg test antibody conjugates h1702-3024, h1702DS-cys-3024, and h1704-3-cys-3024 to rats, the total ADC (total ADC) half-life in the rat is about 97h ( 4.04 days), 98h (4.08 days), 65h (2.71 days).

Test Example 7. Physical stability of B7H3 antibody

DSC was used to detect the thermal stability of different antibodies, and the thermal stability of different buffer systems at different pH conditions was compared. Exemplary buffer systems corresponding to different pHs were 10 mM PB (pH 7), 10 mM Acetate (pH 5.2). The sample was replaced in the corresponding buffer, and the concentration of the sample was adjusted to about 1 mg / ml, and the detection was performed using Micro Cal * VP-Capillary DSC (Malvern). Before testing, degas each sample and blank buffer with a vacuum degasser for 1-2 minutes. Add 400 μl of sample or blank buffer to each well of the sample plate (300 μl on the instrument). Add 14% Decon 90 and ddH ₂ O to the last two pairs of well plates for cleaning. After the sample plate is loaded, put on a soft plastic cover. The scanning temperature is from 25 ° C to 100 ° C, and the scanning rate is 60 ° C / h. The specific results are shown in Table 7. In several test systems, h1702 and h1703 all showed good thermal stability.

SEC-HPLC monitors sample purity to examine the periodic stability at a certain concentration. Exemplary conditions such as adjusting the sample concentration to about 40-50 mg / ml, in a PBS (pH7.4) system and pH5.2 acetic acid / In the sucrose system, the stability of different antibodies at repeated freeze-thaw cycles such as -80 ° C and storage at 4 ° C, 30 ° C, and 40 ° C for one month was compared. The Xbridge protein BEH SE C200 A (Waters) HPLC column was used to check the purity of the antibodies. After a month's investigation, h1702 and h1703 showed good stability. The results are shown in Table 8.

The results showed that h1702 and h1703 showed good cycle stability in both acetic acid and PBS buffer.

Test Example 8. Chemical stability of B7H3 antibody

Chemical modification of antibodies after preparation is one of the common problems that lead to product stability. In particular, some amino acids in the CDR regions are highly deamidated, oxidized, or isomerized. Generally, they are selected to avoid or reduce mutations as much as possible. Take 500 μg of the test antibody in 500 μl of 7.4 pH PBS in a 40 ° C water bath; take samples at 0, 10, and 20 days for enzymatic hydrolysis experiments. Dissolve 100 μg samples taken at different time points in 100 μl 0.2M His-HCl, 8M Gμa-HCl, pH 6.0 solution, add 3 μl 0.1g / mL DTT, and bath in water at 50 ° C for 1 hour, then use 0.02M His-HCl, The pH 6.0 solution was ultra-filtered twice, 3 μL of 0.25 mg / mL trypsin was added, and the solution was hydrolyzed at 37 ° C. overnight. The Agilent 6530Q-TOF was analyzed by LC-MS, and the potential modification sites were analyzed by mass spectrometry (see Table 9 for the results). The results show that no h1702, h1703 involved in the present invention have no significant deamidation, oxidation or isomerization. Aggravating the trend, suggesting that the molecule has good physical and chemical stability.

Test example 9: Cell proliferation experiment

In this experiment, the inhibitory effect of B7H3-ADC on the proliferation of U87MG cells and ZR-75-1 cells was evaluated based on the IC50 size by measuring the intracellular ATP content.

1.Inhibitory effect on U87MG cell proliferation

U87MG cells (Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138) were cultured in EMEM medium containing 10% FBS, and were subcultured 2 to 3 times a week, and the subcultures were listed as 1: 2 or 1: 5. During subculture, aspirate the medium, rinse the cell layer with 5ml 0.25% trypsin, then suck the trypsin, put the cells in the incubator for 3 to 5 minutes, and resuspend the cells by adding fresh medium. 90 μL of cell suspension was added to a 96-well cell culture plate with a density of 4 × 10 ⁴ cells / ml, and the medium was 10% FBS DMEM. Only 100 μl of 10% FBS DMEM medium was added to the periphery of the 96-well plate. The plate was cultured in an incubator for 24 hours (37 ° C, 5% CO ₂ ).

The sample to be tested was diluted to 50 mM with PBS or DMSO, and then diluted 3 times to 10 concentrations, and set as blank and control wells. 10 μl of the compound solution prepared in a gradient concentration was added to 90 μl of fresh medium. 10 μl of the above-mentioned drug-containing medium solution was added to the culture plate. The plates were incubated for 3 days in an incubator _{(37 ℃, 5% CO 2} ). In a 96-well cell culture plate, add 100 μl of Cell Titer-Glo reagent to each well, and store at room temperature in the dark for 5-10 min. Read the chemiluminescence signal value in Victor3. The data is processed using Graph Pad software. The measured IC50 values are shown in Table 10.

CONCLUSION: ADCs obtained from naked anti-conjugated toxins have a good killing effect on U87MG cells.

2.Inhibition effect on ZR-75-1 cell proliferation

ZR-75-1 cells (ATCC, Catalog # CRL-1500) were cultured in RPMI-1640 medium containing 10% FBS, and were subcultured 2 to 3 times a week. The subcultures were listed as 1: 4 or 1: 6. During subculture, aspirate the medium, rinse the cell layer with 5mL of 0.25% trypsin, and then aspirate the trypsin. Place the cells in the incubator and digest for 3 ~ 5 minutes. Add fresh medium to resuspend the cells. 90 μL of cell suspension was added to a 96-well cell culture plate with a density of 2.8 × 10 ⁴ cells / mL. Only 100 μL of 10% FBS RPMI-1640 medium was added to the periphery of the 96-well plate. The plates were incubated for 24 hours in an incubator _{(37 ℃, 5% CO 2} ).

The sample to be tested was diluted to 50 mM with PBS or DMSO, and then diluted 3 times to 10 concentrations, and set as blank and control wells. 10 μl of the compound solution prepared in a gradient concentration was added to 90 μl of fresh medium. 10 μl of the above-mentioned drug-containing medium solution was added to the culture plate. The plates were incubated for 6 days in an incubator _{(37 ℃, 5% CO 2} ). In a 96-well cell culture plate, add 100 μL of Cell Titer-Glo reagent to each well, and place at room temperature in the dark for 5-10 min. Read the chemiluminescence signal value in Victor3. The data is processed using Graph Pad software. The measured IC50 values are shown in Table 11.

CONCLUSION: The ADC obtained by naked anti-conjugated toxin has good killing effect on ZR-75-1 cells.

3. Inhibitory effect on the proliferation of Detroit 562 cells

Detroit 562 cells (ATCC, Catalog # CCL-138) were cultured in EMEM medium containing 10% FBS, and they were subcultured 2 to 3 times a week, and the subcultures were listed as 1: 4 or 1: 6. During subculture, aspirate the medium, rinse the cell layer with 5mL of 0.25% trypsin, and then aspirate the trypsin. Place the cells in the incubator and digest for 3 ~ 5 minutes. Add fresh medium to resuspend the cells. 90 μL of cell suspension was added to a 96-well cell culture plate with a density of 2.2 × 10 ⁴ cells / mL, and only 100 μL of 10% FBS EMEM medium was added to the periphery of the 96-well plate. The plates were incubated for 24 hours in an incubator _{(37 ℃, 5% CO 2} ).

The sample to be tested was diluted to 50 mM with PBS or DMSO, and then diluted 3 times to 10 concentrations, and set as blank and control wells. 10 μl of the compound solution prepared in a gradient concentration was added to 90 μl of fresh medium. 10 μl of the above-mentioned drug-containing medium solution was added to the culture plate. The plates were incubated for 6 days in an incubator _{(37 ℃, 5% CO 2} ). In a 96-well cell culture plate, add 100 μL of Cell Titer-Glo reagent to each well, and place at room temperature in the dark for 5-10 min. Read the chemiluminescence signal value in Victor3. The data is processed using Graph Pad software. See Table 12 for the measured IC50 values.

CONCLUSION: ADCs obtained from naked anti-conjugated toxins have a good killing effect on Detroit 562 cells.

4. Test h1702DS, h1704-3 antibodies and their corresponding ADCs as described above: h1702-cys-3024, h1702DS-cys-3024, h1704-3-cys-3024 Test U87MG, ZR-75-1 and Detroit 562 The cell proliferation inhibitory effect is shown in Table 13 below:

CONCLUSION: ADCs obtained by naked anti-conjugated toxin have good killing effect on U87MG, ZR-75-1 and Detroit 562 cells.

Biological evaluation of in vivo activity

Test Example 10: Efficacy Evaluation of ADC on Human Brain Astroblastoma U87MG Nude Mice Transplantation Tumor

First, the test method

Experimental BALB / cA-nude nude mice, female, 6-7 weeks, were purchased from Shanghai Xipuer. Becky Laboratory Animal Co., Ltd. (Certificate number: SCXK (Shanghai) 2008-0016). Rearing environment: SPF level. Nude mice were subcutaneously inoculated with human brain astrocyte tumor U87MG cells (Chinese Academy of Sciences, Cat. No. TCHu138). On the tenth day after the cells were inoculated (the average tumor volume was 122 mm ³ ), the animals were randomly divided into groups (D0), 8 in each group. Intraperitoneal injection was administered once / week for a total of 3 times. Tumor volume and body weight were measured 2-3 times a week, and data were recorded. Tumor volume (V) is calculated as: V = 1/2 × a × b2

Where a and b represent length and width, respectively.

Relative volume (RTV) = VT / V0

Tumor inhibition rate (%) = (CRTV-TRTV) / CRTV (%)

V0 and VT are the tumor volume at the beginning and end of the experiment, respectively. CRTV and TRTV are the relative tumor volume of the control group (blank) and the experimental group at the end of the experiment.

Test object

h1702-3024ADC (1mpk, 3mpk, 10mpk); h1702DS-cys-3024ADC (1mpk, 3mpk); h1704-3-cys-3024ADC (1mpk, 3mpk); blank group (blank): PBS buffer at pH7.4

Antitumor effect of antibody ADC

1) On the 26th day (D26) after the start of administration, the tumor inhibition rates of the test antibody h1702-3024ADC (1mpk, 3mpk, 10mpk) were 33.68%, 99.13%, and 100%, respectively; see Figure 5, and Compared with the control group, there were significant differences. On the 21st day of administration, due to the tumors being too large in the control group, the weight of individual mice decreased significantly, and on the 26th day of administration, two mice in the control group died due to the tumors being too large. All other groups were normal in weight, see Figure 6, Table 14, and no mice died during the administration, suggesting that there was no significant toxic and side effect of each dose of h1702-3024.

There is a certain dose-dependent relationship between the various doses of h1702-3024. At 3mpk, it has reached a tumor inhibition rate of more than 95%, and 10mpk has reached a tumor inhibition rate of 100%.

2) The experimental results of the tumor suppressive effects of the test antibodies h1702DS-cys-3024ADC and h1704-3-cys-3024ADC are shown in Table 15. The results showed that intraperitoneal injection was administered once / week for a total of 3 times. When D26 was observed, the tumor inhibition rate of the tested ADCh1702DS-cys-3024 (1mpk) reached 22.99%; h1702DS-cys-3024 (3mpk) The tumor inhibition rate reached 98.16%; the tumor inhibition rate of h1704-3-cys-3024 (1mpk) reached 31.05%; the tumor inhibition rate of h1704-3-cys-3024 (3mpk) reached 94.83%; 3 test antibodies of 1mpk There was no significant difference between the group and the blank group (P> 0.05), and the 3 test antibody groups at 3mpk had significant differences compared with the blank group (P <0.001).

During the administration, the weight of the animals in each group was normal, suggesting that ADC had no obvious toxic and side effects.

The tumor volume in the blank group and the 1mpk group was larger at 26 days. Therefore, the blank and 1mpk groups were sacrificed at D26. When the remaining groups were observed to D36, the tumor growth of the h1702DS-cys-3024 (3mpk) group was still slow after stopping treatment , The best antitumor effect. There was no significant difference between h1702DS-cys-3024 (3mpk) and h1704-3-cys-3024 (3mpk) in the tumor inhibition rates between D26 and D36 (P> 0.05).

Test Example 11: Efficacy Evaluation of ADC on Human Pharyngeal Carcinoma Pleural Effusion Metastatic Cells Detroit562 Nude Mice Transplantation Tumors

First, the test method

In the experiment, nude mice BALB / cA-nude, female, 6-7 weeks, were subcutaneously inoculated with human pharyngeal carcinoma pleural fluid metastasis cells Detroit562 cells. On the tenth day after the cells were inoculated, the animals were randomly divided into groups (D0), with 8 animals in each group. Intraperitoneal injection was administered once / week for a total of 3 times. Tumor volume and body weight were measured 2-3 times a week, and data were recorded. . Tumor volume (V) is calculated as: V = 1/2 × a × b2

Where a and b represent length and width, respectively.

Relative volume (RTV) = VT / V0

Tumor inhibition rate (%) = (CRTV-TRTV) / CRTV (%)

V0 and VT are the tumor volume at the beginning and end of the experiment, respectively. CRTV and TRTV are the relative tumor volume of the control group (blank) and the experimental group at the end of the experiment.

Test object

h1702DS-cys-3024ADC (1mpk, 3mpk); blank group (blank): PBS buffer at pH 7.4

Antitumor effect of antibody ADC

When D35 was observed, the tumor suppression rates of the tested antibody ADCs were: h1702DS-cys-3024ADC (1mpk) tumor inhibition rate reached 39.22% (P <0.05); h1702DS-cys-3024ADC (3mpk) tumor inhibition rate reached 70.50 % (P <0.001) (see Table 16). Animals in each group had normal body weight during the medication.

    Evaluation of stability    

Test Example 12: Physical stability of B7H3ADC

DSC was used to detect the thermal stability of ADCs of different antibodies. The thermal stability of different buffer systems at different pH conditions was compared. Exemplary buffer systems corresponding to different pHs were 10 mM PBS (pH 7.4), 10 mM Acetate (pH 5.5). ). The sample was replaced in the corresponding buffer, and the concentration of the sample was adjusted to about 1 mg / ml, and the detection was performed using Micro Cal * VP-Capillary DSC (Malvern). Before testing, degas each sample and blank buffer with a vacuum degasser for 1-2 minutes. Add 400 μl of sample or blank buffer to each well of the sample plate (300 μl on the instrument). Add 14% Decon 90 and ddH ₂ O to the last two pairs of well plates for cleaning. After the sample plate is loaded, put on a soft plastic cover. The scanning temperature is from 25 ° C to 100 ° C, and the scanning rate is 60 ° C / h. The specific results are shown in Table 17. In several test systems, h1702-3024 and h1703-3024 all showed good thermal stability.

Test example 13: ADC stability

SEC-HPLC monitors the purity of samples (h1702DS-cys-3024ADC, h1704-3-cys-3024ADC) to investigate the stability under certain concentration conditions. Exemplary conditions such as regulating the sample concentration to about 50mg / ml, .4) Comparison of the stability of different antibodies stored at 40 ° C for 28 days in the system and pH5.5 acetic acid / sucrose system (referred to as the 559 system). The Xbridge protein BEH SE C200A (Waters) HPLC column was used to check the purity of the antibody. After 28 days of investigation, h1702DS-cys-3024ADC showed good stability. The results are shown in Figure 7.

The results showed that h1702DS-cys-3024 showed good stability in both acetic acid and PBS buffer.

Although the above invention has been described in detail with the aid of drawings and examples for the sake of clear understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

<110> Jiangsu Hengrui Pharmaceutical Co., Ltd., Shanghai Hengrui Pharmaceutical Co., Ltd.

<120> B7H3 antibody-drug conjugate and its medical use

<130> 370063CG

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<223> h1704VH2

<400> 37

<210> 38

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> h1704VL1

<400> 38

<210> 39

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> h1704VL2

<400> 39

<210> 40

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> h1704-3 antibody heavy chain sequence

<400> 40

<210> 41

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> h1704-3 antibody light chain sequence

<400> 41

Claims (27)

An antibody-drug conjugate represented by the general formula (A), Ab- (L ₂ -L ₁ -D) _y (A) wherein: D is a cytotoxic drug; L ₁ and L ₂ are linker units; y is A number selected from 1 to 8; Ab is a B7H3 antibody or an antigen-binding fragment thereof that binds to human B7H3, and the B7H3 antibody or an antigen-binding fragment thereof is a monoclonal antibody selected from any one of (a) to (c) Or an antigen-binding fragment thereof: (a) a monoclonal antibody comprising one or more CDR sequence sequences selected from the following or an amino acid sequence having at least 95% sequence identity with the sequence: an antibody heavy chain variable region HCDR region Sequence: as shown in SEQ ID NO: 10, 11 and 12 amino acid sequences; and antibody light chain variable region LCDR region sequence: as shown in SEQ ID NO: 13, 14 and 15 amino acid sequences; (b) A monoclonal antibody comprising one or more CDR sequence sequences selected from the following or an amino acid sequence having at least 95% sequence identity with it: the antibody heavy chain variable region HCDR region sequence: such as SEQ ID NO: 16, 17 and 18 amino acid sequences; and the antibody light chain variable region LCDR region sequence: as shown in SEQ ID NOs: 19, 20, and 21 amino acid sequences; (c) a monoclonal antibody comprising 1 or Multiple selected from CDR region sequence or amino acid sequence having at least 95% sequence identity with it: the antibody heavy chain variable region HCDR region sequence: as shown in SEQ ID NOs: 30, 31, and 32 amino acid sequences; and the antibody light chain Variable region LCDR region sequence: as shown in SEQ ID NOs: 33, 34 and 35 amino acid sequences. An antibody-drug conjugate represented by the general formula (A), Ab- (L ₂ -L ₁ -D) _y (A) wherein: D is a cytotoxic drug; L1 and L2 are linker units; y is selected from Numbers from 1 to 8; Ab is a monoclonal antibody or antigen-binding fragment thereof that competes with the B7H3 antibody or antigen-binding fragment thereof described in item 1 of the scope of application for binding to human B7H3.     The antibody-drug conjugate according to claim 1 or 2, wherein y is a number selected from 2 to 4.         The antibody-drug conjugate according to any one of claims 1 to 3, wherein the Ab is a recombinant antibody.         The antibody-drug conjugate according to item 4 of the scope of the patent application, wherein the constant region and / or framework region of the Ab is a human-derived recombinant antibody or an antigen-binding fragment thereof.         The antibody-drug conjugate according to item 5 of the scope of patent application, wherein the sequences of the light chain FR region and the heavy chain FR region on the light and heavy chain variable regions of the Ab are derived from the human germline light chain, respectively. And heavy chain sequences or mutant sequences thereof; wherein the constant region includes heavy chain constant regions derived from human IgG1, IgG2, IgG3 or IgG4 or variants thereof; Chain constant region.         The antibody-drug conjugate according to item 6 of the application, wherein the constant region includes a constant region derived from a human IgG1 heavy chain.         The antibody-drug conjugate according to item 6 or 7 of the scope of application for a patent, wherein the Ab contains the antibody represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or SEQ ID NO: 37. Heavy chain variable region or a variant thereof; the variant has a 1 on the heavy chain variable region sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36, or SEQ ID NO: 37 To 10 amino acid substitution sequences; and a light chain variable region or a variant thereof comprising SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38, or SEQ ID NO: 39; The body is a sequence having 1 to 10 amino acid substitutions on the light chain variable region sequence shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 38, or SEQ ID NO: 39.         The antibody-drug conjugate according to item 6 of the patent application scope, wherein the Ab contains a monoclonal antibody or an antigen-binding fragment thereof selected from any one of the following (1) to (7): (1) a single strain An antibody comprising the heavy chain variable region of the antibody shown in SEQ ID NO: 6; and an antibody light chain variable region shown in SEQ ID NO: 7; (2) a monoclonal antibody comprising the sequence shown in SEQ ID NO: 8 An antibody heavy chain variable region; and an antibody light chain variable region shown in SEQ ID NO: 9; (3) a monoclonal antibody comprising the antibody heavy chain variable region shown in SEQ ID NO: 28; and SEQ ID The variable region of the light chain of the antibody represented by NO: 29; (4) a monoclonal antibody comprising the variable region of the heavy chain of the antibody represented by SEQ ID NO: 36; and the variable region of the light chain of the antibody represented by SEQ ID NO: 38 (5) a monoclonal antibody comprising the heavy chain variable region of the antibody shown in SEQ ID NO: 36; and a light chain variable region of the antibody shown in SEQ ID NO: 39; (6) a monoclonal antibody comprising the SEQ The variable region of the heavy chain of the antibody shown in ID NO: 37; and the variable region of the light chain of the antibody shown in SEQ ID NO: 38; (7) a monoclonal antibody comprising the heavy chain of the antibody shown in SEQ ID NO: 37; Variable region; and the light chain variable region of the antibody shown in SEQ ID NO: 39         The antibody-drug conjugate according to any one of claims 1 to 9, wherein the Ab is a full-length antibody, which further includes a human antibody constant region; wherein the full-length antibody is selected from the group consisting of an h1702 antibody, Is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO: 22 and the light chain sequence shown in SEQ ID NO: 23, h1703 antibody, which is a heavy chain sequence shown in SEQ ID NO: 24 and SEQ ID A full-length antibody composed of the light chain sequence shown in NO: 25, h1702-DS antibody, which is a full-length antibody composed of the heavy chain sequence shown in SEQ ID NO: 22 and the light chain sequence shown in SEQ ID NO: 26, And h1704-3 antibody, which is a full-length antibody consisting of the heavy chain sequence shown in SEQ ID NO: 40 and the light chain sequence shown in SEQ ID NO: 41.         The antibody-drug conjugate according to any one of claims 1 to 9, wherein the antigen-binding fragment is selected from Fab, Fab ', F (ab') 2, single-chain antibody (scFv), Dimerized V region (diabody), disulfide stabilized V region (dsFv), and antigen-binding fragments of CDR-containing peptides.         The antibody-drug conjugate according to any one of claims 1 to 11, wherein the cytotoxic drug is selected from the group consisting of a toxin, a chemotherapeutic drug, an antibiotic, a radioisotope, and a nucleolytic enzyme.         The antibody-drug conjugate according to item 12 of the patent application scope, wherein the cytotoxic drug is selected from the group consisting of DM1, DM3, DM4, MMAF and MMAE.     The antibody-drug conjugate according to item 12 of the application, wherein the cytotoxic drug is selected from: The antibody-drug conjugate according to item 12 of the scope of patent application, which is a compound represented by Formula I or a pharmaceutically acceptable salt or solvent compound thereof, Wherein: L ₁ and L ₂ are linker units; y is a number selected from 1 to 8; Ab is the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 of the scope of patent application.     The antibody-drug conjugate according to item 15 of the scope of application, wherein y is a number selected from 2 to 4.     An antibody as defined in claim 15. Item range - drug conjugate, wherein, L ₂ as shown in the following formula L _2: Wherein X _{1 is} selected from a hydrogen atom, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl; X _{2 is} selected from alkyl, cycloalkyl and heterocyclic; m is an integer selected from 0 to 5 ; S is a sulfur atom.     The antibody-drug conjugate according to item 17 of the application, wherein m is 1, 2 or 3.     The antibody-drug conjugate according to item 17 of the scope of patent application, wherein L _{1 is represented by the} following general formula (L ₁ ): Wherein X ₃ is an alkyl group, the alkyl group is further substituted with a substituent selected from halogen, hydroxy, and cyano if necessary; n is an integer selected from 0 to 5.     The antibody-drug conjugate according to item 19 of the application, wherein n is 1, 2 or 3.     The antibody-drug conjugate according to item 15 of the patent application scope, which is an antibody-drug conjugate represented by the general formula (II): The antibody-drug conjugate according to item 15 of the patent application scope, which is an antibody-drug conjugate represented by the general formula (III): The antibody-drug conjugate according to item 1 or 2 of the patent application scope, which is selected from the following compounds:     A pharmaceutical composition comprising the antibody-drug conjugate or the pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate according to any one of claims 1 to 23 of the scope of patent application, and one or A variety of pharmaceutically acceptable excipients, diluents or carriers.         An antibody-drug conjugate according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24, which is used for preparing human B7H3 related Drugs for the disease.         The use as described in item 25 of the scope of patent application, which is used for preparing a medicament for treating B7H3 overexpressing cancer.         An antibody-drug conjugate according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24, which is used for preparing a medicament for treating a disease. The disease is selected from the group consisting of human brain astrocyte tumor, human pharyngeal carcinoma, adrenal tumor, AIDS-related cancer, acinar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cancer, metastatic brain tumor , Breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative small round cell tumor, ependymal cell tumor , Juventus tumor, extra-bone myxoid chondrosarcoma, bone fiber hypoplasia, osteofibrosis, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, card Posey's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipoma tumor, liver cancer, lymphoma, lung cancer, neuroblastoma, melanoma, meningiomas, multiple endocrine neoplasia, multiple Primary myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannomas, pheochromocytoma, Pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, Metastatic thyroid and uterine cancer.